The long-term objective of this project is to develop novel homing endonucleases that can be used in gene targeting protocols to repair the genetic mutations that cause cancer and other human diseases. Homing endonucleases initiate gene targeting by generating double-strand breaks at defined genomic loci that greatly stimulate DNA repair through homologous recombination. The I-Scel protein from S. cerevisiae is a member of the LAGLIDADG family of homing endonucleases that is capable of finding and cleaving a single 18 base-pair recognition sequence within several megabases of non-specific DNA. It has become the reagent-of-choice for studying DNA repair and for initiating gene targeting because of its extreme specificity, which limits DNA cleavage at ectopic targets that negatively impact cell viability. This proposal expands the utility of I-Scel by more precisely controlling its activity in vivo and by evolving the enzyme to specifically recognize other DNA targets. The recent determination of the X-ray structure of I-Scel reveals a two-domain topology that permits the re-engineering of the enzyme. The first Specific Aim engineers molecular switches into the enzyme that allow the activity to be temporally and spatially regulated. Fusion of the I-Scel domains to different protein partners will create one endonuclease whose activity is triggered by the small-molecule rapamycin and one that can be reversibly switched on and off in response to light. The generation of these enzymes will permit the first in vivo regulation of the DNA repair activity and will allow repair events to be studied in single cells. The second Specific Aim uses a bacterial two-hybrid method to alter the DNA target specificity of I-Scel. Individual variants will be selected from a large randomized I-Scel expression library that gain the ability to bind to a mutant recognition sequence but lose the wild-type target specificity. These studies will ultimately increase the repertoire of homing endonucleases available for gene targeting.